GLP-1 induces numerous biological effects such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, inhibiting gastric motility or intestinal motility, enhancing glucose utilization, and inducing weight loss. GLP-1 may further act to prevent the pancreatic β-cell deterioration that occurs as non-insulin dependent diabetes mellitus (NIDDM) progresses. A significant characteristic of GLP-1 is its ability to stimulate insulin secretion without the associated risk of hypoglycemia. GLP-1 induces insulin secretion only when glucose levels are elevated unlike other therapies that act by increasing insulin expression regardless of whether glucose levels are elevated.
The usefulness of therapy involving GLP-1 peptides has been limited by the fact that GLP-1(1-37) is poorly active, and the two naturally occurring truncated peptides, GLP-1(7-37)OH and GLP-1(7-36)NH2, are rapidly cleared in vivo and have extremely short in vivo half lives. It is known that endogenously produced dipeptidyl-peptidase IV (DPP-IV) inactivates circulating GLP-1 peptides by removing the N-terminal histidine and alanine residues and is a major reason for the short in vivo half-life.
While various approaches have resulted in GLP-1 compounds with a longer half-life or greater potency than that of native GLP-1, additional compounds are needed to further decrease clearance and increase half-life thereby optimizing GLP-1's ability to be useful as a therapeutic that can be administered a minimum number of times during a prolonged period of time. International Application Nos. PCT/US2004/006082 and PCT/US2000/11814 describe covalent attachment of one or more molecules of PEG to various GLP-1 and exendin compounds. These compounds may have a half-life in excess of 24 hours allowing for fewer administrations of the PEGylated GLP-1 compound while maintaining a high blood level of the compound over a prolonged period of time.
Further research has elucidated a problem wherein the separation of PEG from a PEGylated GLP-1 or exendin compound occurs during prolonged shelf storage. As a result, the free GLP-1 or exendin peptide increases the initial peak concentration exposure profile through the therapeutic window. This has the possibility of increasing the side effects of nausea and vomiting.
The present invention seeks to overcome the problems associated with the prolonged shelf storage and the potential of separation of PEG from the PEGylated GLP-1 or exendin compound by introducing two PEGylation sites into a GLP-1 or exendin compound and then PEGylating those two PEGylation sites simultaneously. The advantages of this approach are at least four-fold. First, PEGylation of the compounds will dramatically improve the in vivo half-lives of the compounds. Second, PEGylation of the compounds will slow down the absorption rate of the compound and thus, reduce initial burst of the drug that is believed to be responsible for the side effects. Third, linear PEGs can be used directly for PEGylation and will simplify the synthesis procedure. Fourth, tandem PEGylation will alleviate the issues associated with prolonged shelf storage and the potential of separation of PEG from the PEGylated GLP-1 or exendin compound by decreasing the probability that both PEGs will be separated from the same GLP-1 or exendin peptide molecule.
Additionally, introducing two PEGylation sites into a GLP-1 or exendin compound at the C-terminal end of the compound and then PEGylating those two PEGylation sites simultaneously resulted in PEGylated compounds having greater activity over those PEGylated compounds wherein at least one of the PEGylation sites is not at the C-terminal end of the peptide. Further, attaching a linker comprising two PEGylation sites at the C-terminal end of a GLP-1 compound and then PEGylating those two PEGylation sites simultaneously resulted in PEGylated GLP-1 compounds having greater activity over those PEGylated compounds wherein the PEGylation sites are attached at the C-terminal end of a GLP-1 compound without a linker.
Such PEGylated GLP-1 compounds may be used therapeutically to treat subjects with disorders including, but not limited to, diabetes, obesity, gastric and/or intestinal motility abnormalities, beta (β) cell deficiency (e.g. insufficient or nonfunctioning β cells), and gastric and/or intestinal emptying abnormalities with a particular advantage being that the PEGylated GLP-1 compounds of the invention require fewer doses during a 24 hour period, increasing both the convenience to a subject in need of such therapy and the likelihood of subject's compliance with dosing requirements.